Signal peptide and application thereof in synthesis of l-arginine from conjac powder and value enhancement of conjac powder

ABSTRACT

The present invention relates to application of a novel signal peptide in L-arginine and its derivatives production from konjac powder, which belongs to the field of gene engineering, enzyme engineering and metabolism engineering. The present invention fused the signal peptide set forth in SEQ ID NO.1 with the β-mannanase of Bacillus subtilis CCTCC M 209200, and expressed the fused gene in the strain with high L-arginine yield. The recombinant strain Corynebacterium crenatum CGMCC 0890/p MSPman had advantages on utilizing cheaper konjac powder as substrate, and after fermenting for 96 hours in a 5 L bioreactor, the L-arginine yield reached 45 g/L. Another two recombinant strains were constructed based on Corynebacterium crenatum CGMCC 0890/pMSPman, and after fermenting for 96 hours in a 5 L bioreactor, the L-ornithine yield and L-citrulline reached 23.5 g/L and 26.3 g/L respectively.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to application of a novel signal peptide inL-arginine and its derivatives production from konjac powder, whichbelongs to the field of gene engineering, enzyme engineering andmetabolism engineering.

Description of the Related Art

Konjac powder, which made from non-grain crops konjac, is rich in konjacmannan. Konjac is mainly distributed in China, Japan, Burma, Vietnam,Indonesia, Southeast Asia and Africa and other regions. Konjac, whichbelongs to Araceae, is rich in resources, and can be intercropped withfood crops, or grown in silt or beaches, thus it doesn't compete arableland with crops, and can be further developed. According to relatedreports, Yunnan, Guizhou, Sichuan, Hubei and Hunan, Shanxi and otherplaces in the West China are suitable for cultivation of Konjac, therehave been certain cultivation scale, and the cultivation area of konjachave reached 410,000 acres in Hubei province. Currently, konjac powderis mainly used for part of food, and its hydrolyzed oligosaccharides canbe used as functional food, and its application has not been extensivelydeveloped. Konjac, as a main dietary fiber containing food, isidentified as one of the top ten health foods by WHO. However, humancannot digest glucomannan from konjac powder, so its daily intake isless than 10 g. A large surplus of konjac can be used for developmentand utilization. Most microorganisms cannot directly utilize konjacpowder, so it has good application prospects to adapt microbes toferment cheaper konjac powder as a carbon source to producehigh-value-added products.

Corynebacterium crenatum CGMCC No. 0890 has high yield of L-arginine,which is obtained by multistage mutagenesis, genetic engineering andmetabolic engineering technology transformation by our lab. ButCorynebacterium crenatum CGMCC No. 0890 could only utilize glucose asthe main carbon source to produce L-arginine, and konjac powder can't beuse as its direct main carbon source.

L-arginine as a semi-essential amino acid is mainly used in the field ofpharmaceutical industry, health products, food and so on. By means ofgenetic and metabolic engineering technique, the high yield strain canalso ferment konjac powder as carbon source to produce L-arginine. Ithas certain reference application value for simplifying the productionprocess of L-arginine and saving the production cost.

L-arginine can be catalyzed by arginase (EC 3.5.3.1) to produceL-ornithine and urea. L-ornithine as a high value-added product derivedfrom L-arginine is a kind of important non-protein amino acids.L-ornithine can promote the synthesis of protein and the catabolism ofcarbohydrates and lipids, and has an important role in the protection ofliver detoxification function.

L-arginine can be catalyzed by Arginine deiminase (EC 3.5.3.6) toproduce L-citrulline and ammonia. L-citrulline as a high value-addedproduct derived from L-arginine can be used to maintain pulmonaryfunction, improve mental clarity, enhance memory, relax vascular,enhance male sexual function, as well as treat sexual dysfunction.

The strain which can utilize cheap konjac powder as a carbon source toproduce L-arginine is further improved to produce high value-addedL-ornithine and L-citrulline through genetical and metabolic engineeringtechnology, and it has considerable prospects to save food and reduceproduction costs.

DETAILED DESCRIPTION

By taking advantage of genetical engineering, the signal peptide ofβ-mannanase is deleted from itself first and replaced by 12 new onesfrom Sec and Tat secreting pathway from Corynebacterium glutamicum andBacillus subtilis respectively. The effects of signal peptidesreplacement on β-mannanase secretion are compared among differentstrains with high L-arginine production. Finally, the codon of aβ-mannanase signal peptide designated as MSP (Mannase Signal Peptide) isoptimized which is found to be a new signal peptide with a better enzymesecretion ability.

The first goal of the present invention is to provide a signal peptide,comprises one of the characteristics described as following (a) to (d)items:

(a) the nucleotide sequence of it is the same as the sequence set forthin SEQ ID NO. 1;

(b) the nucleotide sequence of it contains the sequence set forth in SEQID NO.1, and the signal peptide has an activity of a Mannase signalpeptide;

(c) the nucleotide sequence of it contains a sequence which has at leastone base of deletion, replacement or addition, compared with thesequence set forth in SEQ ID NO:1; and the signal peptide has anactivity of a Mannase signal peptide;

(d) the nucleotide sequence of it is from Bacillus subtilis, and couldhybridize with the sequence set forth in SEQ ID NO:1 under stringencyconditions; and the signal peptide has a activity of Mannase signalpeptide.

The second purpose of the present invention is to provide a method ofutilizing konjac powder to produce product, the wherein said signalpeptide is fused with β-mannanase, the expression vector of the fusionprotein is introduced to host cell to obtain the recombinant strain, andthen use the recombinant strain ferments the konjac powder as a maincarbon source.

In one embodiment of the present invention, the gene of the wherein saidβ-mannanase is derived from Bacillus subtilis, but not restricted toBacillus subtilis.

In one embodiment of the present invention, the gene of the wherein saidβ-mannanase is derived from Bacillus subtilis CCTCC M 209200, thenucleotide sequence is shown in SEQ ID NO:3.

In one embodiment of the present invention, the wherein said recombinantstrain can produce L-arginine, L-ornithine, or L-citrulline.

In one embodiment of the present invention, the wherein said host strainis Corynebacterium crenatum, but not restricted to Corynebacteriumcrenatum.

In one embodiment of the present invention, the wherein said host strainis Corynebacterium crenatum CGMCC No. 0890.

In one embodiment of the present invention, the wherein said expressionvector is pXMJ19, but not restricted to pXMJ19.

In one embodiment of the present invention, the wherein said recombinantstrain can express arginase gene, the product is L-ornithine.

In one embodiment of the present invention, the wherein said arginasegene derived from Bacillus cereus, but not restricted to Bacilluscereus.

In one embodiment of the present invention, the NCBI number of thewherein said arginase gene is NCBI gi:22934680.

In one embodiment of the present invention, the wherein said recombinantstrain can express arginine deiminase, the product is L-citrulline.

In one embodiment of the present invention, the wherein said arginasedeiminase gene derived from Bacillus cereus, but not restricted toBacillus cereus.

In one embodiment of the present invention, the NCBI number of thewherein said arginase deiminase gene is NCBI gi:22939780.

In one embodiment of the present invention, konjac powder and glucoseare used as mixed carbon source; the effect of glucose is to acceleratethe cell growth during early fermentation phase, konjac powder is usedto induce the secretion of β-mannanase which in turn hydrolyzed konjacpowder to provide effective carbon source to produce related products.

In one embodiment of the present invention, the wherein saidfermentation medium contains 10 g/L konjac powder and 50 g/L glucose,and konjac powder is used as a feed in the late fed-batch fermentationprocess, and the total feed amount of konjac powder is 90 g/L.

The third purpose of the present invention is to provide aCorynebacterium crenatum recombinant strain, the wherein saidrecombinant strain expressed the β-mannanase fused with signal peptidewhose nucleotide sequence is set forth in SEQ ID NO:1; after the whereinsaid signal peptide is fused with β-mannanase, the expression vector ofthe fusion protein is introduced to host cell.

In one embodiment of the present invention, the wherein said recombinantstrain is named of CGMCC 0890/pMSPman, and it is constructed by thefollowing method: β-mannanase of Bacillus subtilis CCTCC M 209200 isfused with the wherein said signal peptide, then gene of the fusedprotein is inserted into pXMJ19 expression vector to form recombinantplasmid named of pXMJ19-MSPman, finally the pXMJ19-MSPman recombinantplasmid is translated to Corynebacterium crenatum CGMCC 0890 to getrecombinant strain CGMCC 0890/pMSPman.

In one embodiment of the present invention, the wherein said recombinantstrain is named of CGMCC 0890/pMSPman-padi, and it is constructed by thefollowing method: connect the padi of Bacillus cereus to the vectorpDXW10, and introduce the expression vector to host cell CGMCC0890/pMSPman.

In the present invention, a new signal peptide which mediated secretionof β-mannanase is invented, and the recombinant strain with this signalpeptide has advantages on utilizing konjac powder to produce relatedproducts, and its utilization efficiency of konjac powder, productionefficiency, and yield are higher than other signal peptides. Therecombinant strain possessing this new signal peptide has advantages onutilizing cheaper konjac powder as substrate to lower the process costson L-arginine and its high value-added products.

In the present invention, based on the strains with high L-arginineyield, the new constructed strain possessing signal peptide invented inthis invention which mediated β-mannanase secretion can utilize mixedcarbon source of konjac powder and glucose to produce L-arginine.Corynebacterium crenatum CGMCC 0890/pMSPman is constructed in thisinvention, after fermenting for 96 hours in a 5 L bioreactor, theL-arginine yield and enzyme activity of 3-mannanase can reach 45±0.9 g/Land 1505±5.7 U/mL respectively. The initial culture medium contains 10g/L konjac powder, 50 g/L glucose, and konjac powder is used as a feedin the later fed-batch fermentation process, and the total feed amountof konjac powder is 90 g/L.

In the present invention, the Corynebacterium crenatum CGMCC0890/pMSPman-pargI which can express arginase is constructed based onCorynebacterium crenatum CGMCC 0890/pMSPman. The Corynebacteriumcrenatum CGMCC 0890/pMSPman-pargI can utilize konjac powder and glucoseas mixed carbon source to produce L-ornithine. The production ofL-ornithine and the enzyme activity of β-mannanase can reach 23.5 g/Land 1610±3.8 U/mL by the fermentation with Corynebacterium crenatumCGMCC 0890/pMSPman-pargI in a 5 L bioreactor for 96 hours after processoptimization. The initial culture medium contains 10 g/L konjac powder,50 g/L glucose, and konjac powder is used as a feed in the laterfed-batch fermentation process, and the total feed amount of konjacpowder is 90 g/L.

In the present invention, the Corynebacterium crenatum CGMCC0890/pMSPman-padi which could express arginine deiminase is constructedbased on Corynebacterium crenatum CGMCC 0890/pMSPman. TheCorynebacterium crenatum CGMCC 0890/pMSPman-padi can utilize konjacpowder and glucose as mixed carbon source to produce L-citrulline. Theproduction of L-citrulline and the enzyme activity of β-mannanase canreach 26.3 g/L and 1360±6.8 U/mL by the fermentation withCorynebacterium crenatum CGMCC 0890/pMSPman-padi in a 5 L bioreactor for96 hours after process optimization. The initial culture medium contains10 g/L konjac powder, 50 g/L glucose, and konjac powder is used as afeed in the later fed-batch fermentation process, and the total feedamount of konjac powder is 90 g/L.

EXAMPLES Material and Methods:

Primers: The fusion primers for the signal peptide and target gene weredesigned according to the related gene sequences published on NCBI.

The strain construction method: The chromosome DNA was extracted fromthe related strains as templates, and the PCR was run according to thepreliminary design of the primers, amplification conditions and system.The PCR products were purified and recovered by using gel extractionkit, and the recovered products concentration were determined by agarosegel electrophoresis of nucleic acid. The related expression vector andpurified PCR products were double-enzyme digested by using the samerestriction endonuclease, the enzyme-digested products were tested byagarose gel electrophoresis and recovered by gel extraction kit, and itsconcentration was determined by ultra-microspectrophotometer. Mixed theenzyme-digested products and the PCR products, and connected them by T4DNA ligase overnight. The recombinant vector was introduced toEscherichia coli BL21 by CaCl₂ transformation method, finallyEscherichia coli BL21 containing recombinant plasmid was constructed.The plasmid was extracted and validated by single and doubleenzyme-digestion and PCR, finally it was introduced to related strain byelectrotransformation method. At last, the recombinant strain was storedat −70° C. with 15% glycerol.

The recombinant strain was inoculated into fresh LB+0.5% Glucose liquidculture medium with 1% inoculum concentration. Then it was transferredto fermentation culture medium which contained konjac powder and glucoseas substrate at next day. The fermentation broth was collected at thelate growth phase and the concentration of amino acids (L-arginine andrelated amino acids) were determined by automatic amino acids analyzer.The fermentation medium contained enough nutritional ingredient that wassuitable for microorganism growth, and had been optimized.

Example 1: the Primers Design for Signal Peptide Tandem β-Mannanase

P1 and P2 for signal peptide tandem β-mannanase were designed accordingto the related gene sequences and β-mannanase gene published on NCBI.The sequences of P1 and P2 were shown in SEQ ID NO:4 and SEQ ID NO:5respectively.

P1: pMSPmanHindIIIF 5′-CCCAAGCTTATGTTCAAGAAGCACACCATCTCCCTGCTGATCATCTTCCTGCTGGCTTCCGCTGTTCTGGCTAAGCCAATCGAGGCTCATACTGTGT CGCCTGTGAATC-3′P2: pMSPmanBamHIR 5′-CGCGGATCCTTACTCAACGATTGGCGTTA-3′

Example 2: Clone and Replacement of Signal Peptide of β-Mannanase

The chromosome DNA was extracted from B. subtilis CCTCC M 209200 astemplates.

The PCR primers for signal peptide tandem β-mannanase were designedaccording to the β-mannanase sequence published on NCBI. MSP (Mannasesignal peptide) tandem β-mannanase was achieved by using the chromosomeDNA of B. subtilis CCTCC M 209200 as template and P1/P2 as primers. Thecodon of MSP was optimized and whose DNA sequence was shown in SEQ IDNO:1, and the sequence of β-mannanase without signal peptide was shownin SEQ ID NO:3. PCR amplification system (50 μL): 1 μL template, 0.5 μLprimers, 4 μL dNTP Mix, 5 μL 10×Ex Taq Buffer, 38.5 μL sterile ddH₂O,0.5 μL Ex Taq DNA polymerase. PCR reaction conditions: Pre-denaturationat 94° C. for 5 minutes for one round; sequential denaturation at 94° C.for 30 seconds, annealing at 56° C. for 30 seconds, and elongation at72° C. for 90 seconds for 30 rounds; elongation at 72° C. for 10 minutesfor one round; 4° C. for 40 minutes for one round. The annealing andelongation temperature should be adjusted according to different primersand genes. PCR products were purified and recovered by gel extractionkit, the purity of products was determined by electrophoresis. Therecovered products were stored in 1.5 mL centrifuge tubes at −20° C.

Example 3: The Construction of Recombinant Vector pXMJ19-MSPman

Joined the recovered PCR products in embodiment 2 and cloning vectorpMD18-T. The ligation system: 5 μL solution, 4.8 μL target gene, 0.2 μLpMD18-T. The ligation between these two DNA molecules was carried out at16° C. overnight. The recombinant plasmid was introduced to E. coilJM109. Then, E.coil JM109 was spread on LB plates containing 100 ug/mLampicillin and cultured at 37° C. overnight. Individual clonal waspicked and inoculated to liquid LB culture medium containing 100 ug/mLampicillin. After cultivation at 37° C. overnight, the plasmid wasextracted outside and named pMD18-T-MSPman. When the validation wassuccessful through PCR and enzyme-digestion, the strain was stored at70° C. with 15% glycerol.

pMD18-T-MSPman and pXMJ19 were cut by HindIII and BamHI, the productswere recovered by gel extraction kit and joined together. Finally,recombinant plasmid called pXMJ19-MSPman was achieved. The validation ofthe recombinant plasmids was successful through PCR andenzyme-digestion.

Example 4: The Electro-Transfection of Corynebacterium crenatum CGMCCNo. 0890 with Recombinant Vector pXMJ19-MSPman

Corynebacterium crenatum CGMCC No. 0890 was picked up and inoculatedinto flasks with 10 mL LB liquid culture medium, and was cultured at 30°C. overnight. 100 μL broth was taken from flask and inoculated intoculture medium for competent cell, and cultured at 30° C. for 4 hoursuntil OD562 reached about 0.9. The broth was centrifuged in steriletubes and the supernatant was discarded. Then the pellets wereresuspended and centrifuged with 10% glycerol for 3 times. Finally, thecompetent cells were divided into 1.5 mL centrifuge tubes equally, and 3uL recombinant plasmid was added into those tubes. The competent cellswith recombinant plasmid were subjected to an electric-transfectionunder the condition of 1800 V and 50 mS. Then the cells were inoculatedinto fresh LBG culture medium (Medium composition: 0.5% yeast extract,1% peptone, 1% NaCl, 0.5% glucose), and placed into water bath at 46° C.for 6 minutes, then cultured at 30° C. for 3 hours. Finally, the cellswere spread on plates containing chloromycetin and cultured at 30° C.for 64 hours. The single colony was picked and cultivated. Then, theplasmid of single colony was extracted and validated throughenzyme-digestion and PCR. The positive recombinant strain was namedCGMCC No. 0890/pMSPman and stored at 70° C.

Example 5: the Determination of Enzyme Activity of β-Mannanase Secretedfrom Recombinant CGMCC No. 0890/pMSPman

2.0 mL 5.0 g/L Locust bean gum substrate solution was added to tubes,then preheated the tubes at 65° C. for 10 minutes. 2.0 mL crude enzymesolution with appropriate concentration was added into tubes, and thereaction was carried out at 65° C. for 10 minutes. 5.0 mL DNS reagentwas added immediately into tubes, then shook those tubes for a while.Place those tubes into boring water for 10 minutes, then use ice waterto cool down tubes. Water was added to make the liquid volume in thetubes to be 10.0 mL, then shook those tubes for a while. 2.0 mL waterwas used to replace the crude enzyme as control group. The absorbance at540 nm was measured to calculate the enzyme activity of β-mannanaseaccording to mannose standard curve. The definition of enzyme activityunit: One unit of enzyme activity of β-mannanase was equal to the enzymeamount needed to produce 1 μmol reducing sugar per minute.

Example 6: Fermentation of Recombinant CGMCC No. 0890/pMSPman

The seed of recombinant CGMCC No. 0890/pMSPman was inoculated to konjacpowder medium under the cultivation condition of 30° C. and 600 r/min.96 hours later, the concentration of L-arginine reached 45±0.9 g/L, andthe enzyme activity of β-mannanase was 1505±5.7 U/mL.

Konjac Powder Medium (g/L):

The initial culture medium contains 10 g/L konjac powder, 50 g/Lglucose, 8 g/L Yeast powder, 20 g/L (NH₄)₂SO₄, 1.5 g/L KH₂PO₄, 0.5 g/LMgSO₄.7H₂O, 0.02 g/L FeSO₄.7H₂O, 0.02 g/L MnSO₄.H₂O, 30 g/L CaCO₃, andthe pH of the liquid medium was between 7.0 to 7.2; After fermenting for12 hours, konjac powder was fed into broth, and the total feed amount ofkonjac powder was 90 g/L.

Control Group:

Similar fermentation process of embodiment 1-6 was employed to cultivatethe control strain which only differed in signal peptide shown in SEQ IDNO:1 that was replaced by the original one of f-mannanase which wasshown in SEQ ID NO:2. The concentration of L-arginine was only 31±3.7g/L, and the enzyme activity of β-mannanase was 1050±3.8 U/mL in controlgroup.

Similar fermentation process of embodiment 1-6 was employed to cultivatethe control strain which only differed in signal peptide shown in SEQ IDNO:1 that was replaced by signal peptide PS (SEQ ID NO:6) of surfaceproteins from Corynebacterium glutamicum or by signal peptide AE (SEQ IDNO:7) of α-amylase from Bacillus subtilis. The concentration ofL-arginine was 26±1.5 g/L and 23±1.2 g/L respectively, and the enzymeactivity of β-mannanase was 867±3.7 U/mL and 732±5.9 U/mL respectivelyin control groups.

To sum up, by taking advantage of signal peptide (SEQ ID NO:1) of thisinvention, the capability of utilizing konjac powder to produce productsof the recombinant strain was significantly improved than by othersignal peptides with 45.2%˜96% increase in production and 43%˜105%increase in the enzyme activity of β-mannanase.

Example 7: The Determination of L-Arginine of Recombinant CGMCC0890/pMSPman

L-arginine in the fermentation broth was first determined by reagentmethod in sakaguchi, Fermented supernatant was diluted 500 times, take 1mL in the colorimetric tube, added 4 mL 0.375 mol/L NaOH solution, thenadded 1 mL of chromogenic agent, shook up and then putted it in 30° C.water for 20 minutes, each tube was measured by spectrophotometer at 520nm absorbance value, according to the standard curve to calculate theL-arginine content in the fermented liquid. Then its accurateconcentration was determined by an amino acids analyzer.

Example 8: the Primer Design for Construction of Recombinant CGMCC0890/pMSPman-PargI

Based on the inventors' former patent (Publication Number:CN103243128A), the designed primer p1 and p2 were shown below in SEQ IDNO:8 and SEQ ID NO:9, respectively.

p1: pargIEcoRIF 5′-CGGAATTCATGAAAAAAGAAATCTCAGTTATTGG-3′ p2: pargISacIR5′-CCGAGCTCTTATTTTAGTTTTTCACCGAATAAA-3′

Example 9: the Construction of Recombinant Plasmid pDXW10-Arg

argI gene containing restriction enzyme cutting site of EcoRI and SacIwas obtained by using Bacillus cereus chromosome as template and primersdesigned in embodiment 8 and purified by related kits. The purified PCRproducts and expression vector pDXW10 were cut by EcoRI and SacI, thenthe products were recovered by gel extraction kit. Join the recoveredproducts at 16° C. overnight. The recombinant plasmid was introduced toE.coil JM109, then spread the cells on the LB plates containing 100ug/mL kanamycin, and cultivated at 37° C. overnight. Single colony waspicked up and inoculated to 10 mL LB liquid culture medium containing100 ug/mL kanamycin. Culture the cells at 37° C. overnight, the extractthe plasmid named pDXW10-argI out of broths. After the validation waspassed through PCR and enzyme-digestion, the strain was stored at −70°C.

Example 10: the Recombinant Plasmid pDXW10-ArgI was Electro-Transfectedto CGMCC 0890/pMSPman

CGMCC 0890/pMSPman was used as an original strain forelectro-transfection, and the antibiotic screening marker was kanamycin.Embodiment 4 was used as a reference. Finally, recombinant strain CGMCC0890/pMSPman-pargI was screened out.

Example 11: The Determination of Enzyme Activity of GlutamateDecarboxylase from Recombinant CGMCC 0890/pMSPman-PargI

Prepare 0.2 M L-arginine substrate solution (pH 9.0, containing 0.2 Mcarbonate buffer solution), then 0.9 ml of substrate solution was mixedwith 0.1 ml of enzyme solution for reaction at 40° C. for 10 minutes.Diluted the enzyme reaction solution to the corresponding times, thentoke 1.0 mL diluted solution to detect the content of L-ornithine withChinard colorimetric method. One enzyme activity unit was equal to theenzyme amount needed for production of 1 umol L-ornithine per minute.

Example 12: the Fermentation of Recombinant CGMCC 0890/pMSPman-PargI

The seed of recombinant CGMCC 0890/pMSPman-pargI was inoculated tokonjac powder medium under the cultivation condition of 30° C. and 600r/min. 96 hours later, the concentration of L-ornithine reached 23.5g/L, and the enzyme activity of β-mannanase was 1610±3.8 U/mL.

Konjac Powder Medium (g/L):

The initial culture medium contains 10 g/L konjac powder, 50 g/Lglucose, 8 g/L Yeast Powder, 20 g/L (NH₄)₂SO₄, 1.5 g/L KH₂PO₄, 0.5 g/LMgSO₄.7H₂O, 0.02 g/L MnSO₄.H₂O, 0.02 g/L FeSO₄.7H₂O, 30 g/L CaCO₃, andthe pH of the liquid medium was between 7.0 to 7.2; After fermenting for12 hours, konjac powder was fed into broth, and the total feed amount ofkonjac powder was 90 g/L.

Control Group:

Similar fermentation process was employed to cultivate the controlstrain which was only differ in signal peptide that shown in SEQ ID NO:1was replaced by the original one of β-mannanase which was shown in SEQID NO:2. The concentration of L-ornithine was 21±0.8 g/L, and the enzymeactivity of 1-mannanase was 1025±5.8 U/mL in control group.

Similar fermentation process was employed to cultivate the controlstrain which was only differ in signal peptide that shown in SEQ ID NO:1was replaced by signal peptide PS (SEQ ID NO:6) of surface proteins fromCorynebacterium glutamicum or by signal peptide AE (SEQ ID NO:7) ofα-amylase from Bacillus subtilis. The concentration of L-ornithine was17±1.5 g/L and 15±1.3 g/L respectively, and the enzyme activity ofβ-mannanase was 865±6.7 U/mL and 752±3.9 U/mL respectively in controlgroups.

To sum up, by taking advantage of signal peptide (SEQ ID NO:1) of thisinvention, the capability of utilizing konjac powder to produceL-ornithine of the recombinant strain was significantly improved thanother signal peptides.

Example 13: The Determination of L-Ornithine from Fermentation Broth ofRecombinant CGMCC 0890/pMSPman-PargI

Amino acid analyzer was used to determine the precise concentration ofL-ornithine from fermentation broth.

Example 14: the Primer Design for Construction of Recombinant CGMCC0890/pMSPman-Padi

Based on the inventors' former patent, the designed primer p3 and p4were shown below in SEQ ID NO:10 and SEQ ID NO:11, respectively.

P3: padiSacIF 5′-CCGAGCTCATGAAGCATCCGATACATGTT-3′ P4: psdiHindIIIR5′-CCCAAGCTTTTAAATATCTTTACGAACAATTG-3′

Example 15: the Construction of Recombinant Plasmid pDXW10-Adi

The primer was shown in example 14, and the operation was shown inexample 9.

Example 16: the Recombinant Plasmid pDXW10-Adi was Electro-Transfectedto CGMCC 0890/pMSPman

CGMCC 0890/pMSPman was used as an original strain forelectro-transfection, and the antibiotic screening marker was kanamycin.Embodiment 4 was used as a reference. Finally, recombinant strain CGMCC0890/pMSPman-padi was screened out.

Example 17: the Determination of Enzyme Activity of GlutamateDecarboxylase from Recombinant CGMCC 0890/pMSPman-Padi

Prepare 0.2 M L-arginine substrate solution (pH 9.0, containing 0.2 Mcarbonate buffer solution), then 0.9 ml of substrate solution was mixedwith 0.1 ml of enzyme solution for reaction at 40° C. for 10 minutes.Diluted the enzyme reaction solution to the corresponding times, thentoke 1.0 mL diluted solution to detect the content of L-citrulline withAmino acid analyzer. One enzyme activity unit was equal to the enzymeamount needed for production of 1 umol L-citrulline per minute.

Example 18: the Fermentation of Recombinant CGMCC 0890/pMSPman-Padi

The seed of recombinant CGMCC 0890/pMSPman-padi was inoculated to konjacpowder medium under the cultivation condition of 30° C. and 600 r/min.96 hours later, the concentration of L-ornithine reached 26.3 g/L, andthe enzyme activity of β-mannanase was 136016.8 U/mL.

Konjac Powder Medium (g/L):

The initial culture medium contains 10 g/L konjac powder, 50 g/Lglucose, 8 g/L Yeast Powder, 20 g/L (NH₄)₂SO₄, 1.5 g/L KH₂PO₄, 0.5 g/LMgSO₄.7H₂O, 0.02 g/L MnSO₄.H₂O, 0.02 g/L FeSO₄.7H₂O, 30 g/L CaCO₃, andthe pH of the liquid medium was between 7.0 to 7.2; After fermenting for12 hours, konjac powder was fed into broth, and the total feed amount ofkonjac powder was 90 g/L.

Control Group:

Similar fermentation process was employed to cultivate the controlstrain which was only differ in signal peptide that shown in SEQ ID NO:1was replaced by the original one of β-mannanase which was shown in SEQID NO:2. The concentration of L-citrulline was 22±0.9 g/L, and theenzyme activity of β-mannanase was 1002±4.7 U/mL in control group.

Similar fermentation process was employed to cultivate the controlstrain which was only differ in signal peptide that shown in SEQ ID NO:1was replaced by signal peptide PS (SEQ ID NO:6) of surface proteins fromCorynebacterium glutamicum or by signal peptide AE (SEQ ID NO:7) ofα-amylase from Bacillus subtilis. The concentration of L-citrulline was19±1.3 g/L and 16±0.7 g/L respectively, and the enzyme activity ofβ-mannanase was 832±7.7 U/mL and 763±6.9 U/mL respectively in controlgroups.

To sum up, by taking advantage of signal peptide (SEQ ID NO:1) of thisinvention, the capability of utilizing konjac powder to produceL-citrulline of the recombinant strain was significantly improved thanother signal peptides.

Example 19: the Determination of L-Ornithine from Fermentation Broth ofRecombinant CGMCC 0890/pMSPman-Padi

Amino acid analyzer was used to determine the precise concentration ofL-citrulline from fermentation broth.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

What is claimed is:
 1. A method of utilizing konjac powder to produce aproduct, comprising making a fusing gene of a signal peptide fused withβ-mannanase, then introducing an expression vector of the fusion gene toa host cell to obtain a recombinant strain, and then producing theproduct from a fermentation culture of the recombinant strain in amedium of konjac powder as a main carbon source; wherein a nucleotidesequence of the signal peptide comprises: (a) a nucleotide sequence setforth in SEQ ID NO:1; or (b) a nucleotide sequence set forth in SEQ IDNO.1, and wherein the nucleotide sequence encodes a Mannase signalpeptide; or (c) a nucleotide sequence with a substitution of at leastone base of deletion, replacement or addition, wherein the substitutionis relative to a parent nucleotide sequence set forth in SEQ ID NO:1;and wherein the nucleotide sequence encodes a Mannase signal peptide; or(d) a nucleotide sequence from Bacillus subtilis, and wherein thenucleotide sequence is capable to hybridize with a sequence set forth inSEQ ID NO:1 under stringency conditions; and wherein the nucleotidesequence encodes a Mannase signal peptide.
 2. The method of claim 1,wherein a gene of the β-mannanase is derived from Bacillus subtilis, oranother strain other than Bacillus subtilis.
 3. The method of claim 1,wherein the product is L-arginine, L-ornithine, or L-citrulline.
 4. Themethod of claim 1, wherein the host cell is Corynebacterium crenatum, oranother host cell other than Corynebacterium crenatum.
 5. The method ofclaim 1, the wherein the expression vector is pXMJ19, or anotherexpression vector other than pXMJ19.
 6. The method of claim 3, whereinthe recombinant strain can express arginase gene, and the product isL-ornithine.
 7. The method of claim 3, wherein the recombinant straincan express arginine deiminase, and the product is L-citrulline.
 8. Themethod of claim 1, wherein the medium comprises 10 g/L konjac powder and50 g/L glucose, and wherein the konjac powder is used as a feed in alater fed-batch fermentation process, and a total feed amount of konjacpowder is 90 g/L.
 9. A recombinant Corynebacterium crenatum, expressinga fusion gene of β-mannanase fused with a signal peptide whosenucleotide sequence is set forth in SEQ ID NO:1; wherein an expressionvector of the fusion gene is introduced to its host cell.
 10. A signalpeptide, wherein a nucleotide sequence of the signal peptide comprises:(a) a nucleotide sequence set forth in SEQ ID NO:1; or (b) a nucleotidesequence set forth in SEQ ID NO:1, and wherein the nucleotide sequenceencodes a Mannase signal peptide; or (c) a nucleotide sequence with asubstitution of at least one base of deletion, replacement or addition,wherein the substitution is relative to a parent nucleotide sequence setforth in SEQ ID NO:1, and wherein the nucleotide sequence encodes aMannase signal peptide; or (d) a nucleotide sequence from Bacillussubtilis, and wherein the nucleotide sequence is capable to hybridizewith a sequence set forth in SEQ ID NO:1 under stringency conditions;and wherein the nucleotide sequence encodes a Mannase signal peptide.